2.1. XENOGRAFT REJECTION
Advances in organ transplantation surgery and the development of effective immunosuppressive drug regimens has made organ transplantation a nearly routine procedure. The shortage of human donor organs is the principal obstacle in the transplantation field. Only a fraction of transplantation candidates receive grafts, and many patients are not even listed as candidates owing to this shortage. In addition, a number of diseases (e.g., diabetes) do not include transplantation as a viable option at this time. However, this perspective could change if transplantation options were more permissive. Accordingly, much attention has recently been placed on alternative animal organ donor sources. Higher primates are immunologically most suitable and have been used as organ donors in a few cases, but are difficult and uneconomical to breed, may impose a high risk of viral transmission and their widescale use in clinical transplantation is likely to raise ethical objections. Consequently, focus has been placed upon use of the pig as an organ donor. Swine constitute an attractive source of organ donors for clinical transplantation because they are plentiful, can be easily bred in captivity, have anatomical and physiologic compatibility with humans and are amenable to genetic manipulation (Cooper et al., 1991, in Xenotransplantation: the transplantation of organs and tissues between species, 481-500 (Springer, Berlin); Tumbleson, M. E. (ed.) 1985, Swine in biomedical Research, Volume 3 (Plenum, N.Y.); Stanton et al. (eds.), 1986 Swine in Cardiovascular Research, Vol. I-III (CRC Press, Florida)).
Transplantation between individuals of the same species or between closely related species is called concordant, and between more distant species, discordant. The management of concordant graft rejection is now possible with immunosuppressive therapy. In contrast, discordant transplantation, such as that between pig and human or old world monkey, is characterized by hyperacute rejection ("HAR"), an extremely rapid immunological attack by preformed host antibodies which recognize molecular structures expressed on the endothelial cell surface of vascularized grafts (Starzl et al., 1993, Lancet 341:65; Auchincloss, H. 1988, Transplantation 46:1; Tuso et al., 1993, Transplantation 56:651; Inverardi et al., 1994, Immunol Rev. 141:71-93). Vascularized grafts performed between discordant species undergo hyperacute rejection within minutes of implant and can lead to graft destruction within approximately 5-20 minutes in the case of a swine to old world monkey transplantation. The mechanisms that mediate hyperacute rejection are not susceptible to conventional immunosuppressive therapy (Auchincloss, H. 1988, Transplantation 46:1). Recent studies have suggested that if HAR is weathered by the transplanted organ, the transplanted organ "accommodates" to the host, and its long-term survival becomes manageable by more conventional immunosuppressive drugs (Platt, J., 1994, Immunol. Rev. 141:127-149; Bach et al., 1991, Transpl. Proc. 23(1):205-207). There is therefore a great need for developing innovative methods and compositions capable of achieving clinically significant prolongation of xenograft function and survival by overcoming hyperacute rejection (Platt et al., 1990, Immunol. Today 11:450).
In swine to old world monkey combinations, the recognition and binding of antigens expressed on the endothelium of the donor organ by preformed xenoreactive IgM antibodies of recipient origin is considered the major immediate mediator of graft endothelial cell injury through complement-dependent hyperacute rejection (Platt et al., 1991, Transplantation 52:214; Dalmasso et al, 1992, Immunopharmacology 24:149). This role of xenoreactive antibodies in the immediate recognition of a xenogeneic organ is suggested by observations that: perfusion of xenogeneic organs results in the selective depletion of xenoreactive natural antibodies from the blood (Perper et al., 1966, Transplantation 4:337-388; Platt et al., 1990, Transplantation 49:1000-1001; Giles et al., 1970, Transplant Proc. 2:522-537; Cooper et al., 1988, J. Heart Transplant 7:238-246; Fischel et al., 1992, J. Heart Lung Transplant 11:965-974; Holzknecht et al., 1995, J. Immunol. 154:4565-4575), depletion of xenoreactive antibodies through perfusion of xenogeneic organs delays hyperacute rejection of a xenograft even when the complement system remains intact (Dalmasso et al., 1992, Am. J. Pathol. 140:1157-1166), hyperacute rejection does not occur when swine hearts are transplanted into newborn old world monkeys which have an intact complement system but very low levels of natural antibodies (Kaplan et al., 1994, Transplantation 59:1-6), infusion of antidonor antibodies may initiate the rejection of a xenogeneic organ graft (Perper et al., 1967, Transplantation 5:514-533; Chavez-Peon et al., 1971, Transplant Proc. 3:573-576) and specific inhibition of the binding of natural antibodies delays the onset of hyperacute rejection (Gamblez et al., 1992, Transplantation 54:577-583; Ye et al., 1994 Transplantation 58:330-337).
The histo-blood group A and B epitopes, against which anti-A and anti-B antibodies are directed, are structurally defined trisaccharides (Lloyd et al., 1968, Biochemistry 7:2976; Watkins, W. M., 1974, Biochem. Soc. Symp. 40:125; Watkins, W. M., 1980, Biochemistry and genetics of the ABO, Lewis and P blood group systems, In: Advances in Human Genetics, Harris and Hirschhorn (eds), Vol. 10, Plenum, New York, p. 1). Baboons "hyperimmunized" to the incompatible donor group through intravenous injection of a composition containing the incompatible donor trisaccharide reject heterotopic allografted ABO-incompatible donor hearts through hyperacute antibody-mediated vascular rejection within a mean of 19 minutes. Continuous intravenous infusion of the incompatible A or B donor group trisaccharide and/or ex vivo depletion with this immobilized trisaccharide, beginning immediately pre-transplantation and continued post-transplantation for several days, has been observed to prolong allograft survival to a mean of 8 days (Cooper et al., 1993, Transplantation, 56:769-777). While these results have led to speculation that the ABO system serves as a model for HAR of xenografts, unlike the group A and B epitopes, the epitope(s) bound by anti-animal antibodies that are determinative of xenograft rejection, have not been structurally characterized thoroughly and effective anti-animal antibody blocking substances have not been described.
Xenoreactive natural antibodies have been shown to play a major role in initiating HAR in the case of old world monkey rejection of a swine xenograft, since their depletion appears to prevent complement activation and abrogates HAR, potentially allowing prolongation of xenograft survival for variable periods (Lu et al., 1994, FASEB, J. 8:1122-1130; Platt et al., 1990, Transplantation 50:817-822). On the other hand, rejection of vascularized discordant xenogeneic organs inevitably takes place after these treatments, suggesting that other mechanisms must be involved in the recognition of the grafts. For example, an induced antibody response may take place, due to sensitization of the recipient (Valvidia et al., 1990, Transplantation 50:132; Monden et al., 1989, Surgery 105:535; Bouwman et al., 1989, Transplant Proc. 21:551; Bouwman et al., Transplant Proc. 21:540; Sachs et al., 1971, J. Immunol. 107:481). Additionally, the alternative pathway for complement activation, which can act in the absence of antibodies, has also been implicated in xenograft rejection and may be capable of at least partially substituting for the direct antibody-dependent pathway (Zhao et al., 1994, Transplantation 57:245; Forty et al., 1993, J. Heart Lung Transpl. 12:283; Wang et al., 1992, Histochem. T. 24:102; Miygawa et al., 1988, Transplantation 46:825; Johnston et al., 1992, Transplantation 54:573). These data suggest that since complement deposition could be observed in a transplanted xenogeneic organ in the absence of Ig deposition, mechanisms leading to rejection may be triggered even if natural xenoreactive antibodies are neutralized or removed from recipient serum.
2.2. .alpha.GAL EPITOPE
In recent years, much attention has been focused on defining the molecular structures that are recognized by xenophilic natural antibodies, leading to activation of the complement cascade and eventually, to hyperacute rejection. Most evidence now points to the oligosaccharide epitope Gal.alpha.1-3Gal (".alpha.Gal") as the major target of xenoreactive natural antibodies. Humans and old world monkeys do not express the .alpha.Gal epitope because they lack a functional gene encoding the enzyme .alpha.1-3galactosyl transferase that forms the unfucosylated "linear B" epitope Gal.alpha.1-3Gal.beta.1-4GlcNAc, which in other mammalian cells causes terminal glycosylation of many glycoproteins, including those expressed by endothelial cells, leukocytes and red blood cells (Galili, et al., 1993, Immunol. Today 14:480-482). Initial evidence of the importance of the .alpha.Gal epitope was provided by studies in which antibodies from porcine organs perfused by human plasma were eluted and tested for binding to immobilized carbohydrates by an ELISA (enzyme-linked immunosorbent assay). Of the carbohydrates tested, the eluted antibodies were observed to bind only to those carbohydrates containing .alpha.-galactose (Good et al., 1992, Transplant Proc. 24:559-562). A subsequent study examining the cytotoxic effect of human and baboon serum on a pig cell line has shown that carbohydrates containing a terminal .alpha.-galactose can neutralize cytotoxicity (Neethling et al., 1994, Transplantation 57:959-963). Additionally, Collins et al., have shown that expression of the Gal.alpha.1-3Gal antigen in donor organs may be sufficient to bring about the immunological reactions leading to hyperacute xenograft rejection and also that removal of Gal.alpha.1-3Gal from porcine cells eliminates the binding of 70-80% of xenoreactive antibody (Collins et al., 1994, Xenotransplantation 1:36-46; Collins et al., 1995, J. Immunol. 154:5500-5510).
Recently, the antigenic glycolipid in pig kidney endothelial cells has been identified as a pentasaccharide consisting of Gal.alpha.1-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4Glc-ceramide (Samuelsson et al., 1994, Immunological Rev. 141:151-168). A study using ELISA and in vitro immunosorbent assays to compare the ability of this .alpha.Gal pentasaccharide to bind human anti-pig antibodies with that of the .alpha.Gal disaccharide (Gal.alpha.1-3Gal) or the .alpha.Gal trisaccharide (Gal.alpha.1-3Gal.beta.1-4GlcNAc) has indicated that human anti-.alpha.Gal antibodies are polymorphic, and that immunoadsorbents containing these .alpha.Gal oligosaccharides may be capable of removing anti-.alpha.Gal activity albeit ineffectively (Goldberg et al., 1995, Transplant Proc. 27:249-250). To date, experiments investigating the ability of oligosaccharides containing the .alpha.Gal epitope to neutralize xenoreactive antibody have been limited to ex vivo hemagglutination, ELISA, and cytotoxicity assays. These limited time frame experiments have demonstrated that the .alpha.Gal disaccharide (Gal.alpha.1-3Gal) and .alpha.Gal trisaccharide (Gal.alpha.1-3Gal.beta.1-4 GlcNAc) are effective in neutralizing the anti-.alpha.Gal antibody in vitro and that the .alpha.Gal trisaccharide is ten times more effective than the .alpha.Gal disaccharide. (Neethling et al., 1996, Transplantation International 9:98-101.)
To date, .alpha.Gal oligosaccharides longer than the .alpha.Gal tetrasaccharide have not been tested individually. Further, none of the Gal.alpha.1-3Gal oligosaccharides have been tested in vivo for the ability to block anti-.alpha.Gal binding. The ex vivo experiments lack the complexity of the organ xenograft system in vivo and therefore do not contain other variables that might participate in the process of rejection in vivo. Additionally, these experiments have been performed using human serum devoid of cells and it is unclear what role respective pathways play in initiation of the hyperacute rejection, but it is likely that no single pathway alone is entirely responsible. For example, the soluble carbohydrate melibiose (Gal.alpha.1-6Glu) is a disaccharide similar structurally to Gal.alpha.1-3Gal that has been shown in vitro to compete with natural .alpha.Gal epitopes for human Ig binding, however, in vivo administration of this composition has failed to prevent hyperacute rejection and has been found to be cytotoxic to other tissue (Ye et al., 1994, Transplantation 58:330-337). Thus in the absence of evidence to the contrary, hemagglutination and cytotoxicity results cannot reasonably be expected to be predictive of successful xenograft engraftment. The key experiment for modeling human xenotransplantation, the grafting of pig organs into old world monkeys, until now, has not been performed.
While scientific data indicates that most human xenoreactive IgM and some human xenoreactive IgG is specific for the Gal.alpha.1-3Gal epitope, some human xenoreactive natural antibodies directed against other determinants may also be responsible for hyperactive rejection, as suggested by Parker et al. (1995, Transpl. Immunol. 3:181-191), Lesnikoski et al. (1995, Xenograft endothelial host-mononuclear cell activation and cytokine expression during rejection of pig to baboon discordant xenografts. Abstracts of the XVth World Congress of the Transplantation Society. Transplantation Proceedings) Ye et al. (1994, Transplantation 58:330; and Collins et al., 1994 Xenotransplantation 1:36). Thus far, Gal.alpha.1-4Gal, Gal.beta.1-3GalNAc, and SO4-3Gal, three other pig carbohydrate specificities to which humans have natural antibodies, have been identified (Holgersson et al., 1990, J. Biochem. 108:766; Holgersson et al., 1991, Glyconj. J. 8:172; and Good et al., 1992, Transplant Proc. 24:559). This possibility is also suggested by the fact that other species such as the pig, goat, dog, rat, etc., which do not produce anti-Gal.alpha.1-3Gal antibodies have xenoreactive natural antibodies which presumably recognize other structures (Cameron et al., 1983, J. Surg. Oncol. 22:157-163; Hammer, C. 1989, 21:522-523). Anti-pig antibodies that bind to the protein components on the surface of pig cells have been reported (Tuso et al., Presentation at the American Society of Transplant Surgeons, 12th Annual Meeting in Houston, May 17-19, 1993). It is possible that antibody dependent and other mechanisms are operating through these epitopes independent of the .alpha.Gal epitope and would be resistant to its inhibitors.
One of the principal concerns of intravenous carbohydrate therapy in xenotransplantation is whether effective xenograft rejection inhibition can be achieved at acceptable non-toxic levels of oligosaccharide. As discussed supra, while ABO-incompatible rejection has been inhibited successfully using intravenous soluble carbohydrates as antibody inhibitors (Cooper et al., 1993, Transplantation 56:769-777), this approach has been unsuccessful in previous pig/primate xenotransplantation where the necessary concentrations of the anti-.alpha.Gal antibody inhibitor melibiose (Gal.alpha.1-6 Glc) proved highly toxic. Anti-.alpha.Gal antibodies are known to bind to .alpha.Gal oligosaccharides with relatively low affinity (Parker et al., 1995 Transplant Immunology 3:181-191; Parker et al., 1994, J. Immunology 153:3791-3803). This low affinity is believed to necessitate a high concentration of oligosaccharide in the recipient's blood in order to block the binding of circulating anti-.alpha.Gal antibodies to the transplanted organ and may result in side effects due to the high concentrations of carbohydrate (see, e.g., U.S. Pat. No. 5,560,911). This low binding affinity is also thought to possibly have an adverse impact on extracorporeal immunoaffinity treatment by making the removal of anti-.alpha.Gal antibodies relatively inefficient (see, e.g., U.S. Pat. No. 5,560,911).
A complicated series of multiple overlapping events contribute to the recognition of vascularized discordant grafts, including binding of preformed natural antibodies, complement activation, activation of the coagulation cascade and endothelial cell activation. Due to these multiple overlapping events, the possible involvement of the antibody-independent alternative pathway, the potential involvement of xenoantibodies other than those specific for the .alpha.Gal epitope, and the low binding affinity of anti-.alpha.Gal antibodies for .alpha.Gal oligosaccharides, one would not reasonably expect that treatment with .alpha.Gal oligosaccharides would be effective in neutralizing anti-.alpha.Gal antibodies and even if they were, that such neutralization and/or removal of anti-.alpha.Gal antibodies would be sufficient to overcome HAR or to attenuate xenograft rejection.
2.3. CURRENT APPROACHES TO OVERCOME XENOGRAFT REJECTION
Other approaches for overcoming HAR are also being explored. These approaches generally aim to genetically engineer pigs so that they do not trigger the rejection reaction, by for example, expressing elevated levels of complement regulatory sequences on the surface of endothelial cells (Langford et al., 1993, Abstract #56, Second International Congress on Xenotransplantation, Cozzi et al., 1993, Abstract #57, Second International Congress on Xenotransplantation), expressing fucosyl transferase that competes with .alpha.-galactosyltransferase for acceptors and fucosylates the acceptor moiety (Sandrin et al., 1996, Xenotranspl. 3:134-140 and Sandrin et al., 1995, Nature Med. 1:1261-1267), or by knocking out the gene encoding .alpha.-galactosyl transferase. An alternative approach attempts to tolerize prospective human recipients to pig tissues, by for example, inducing immunological chimerism (see e.g., Tanaka et al., 1993, Abstract #122, Second International Congress on Xenotransplantation; Zeng et al., 1992, Transpl. Proc. 24:641; Zeng et al., 1992, Transplantation 53:277; Ricordi et al., 1992, Surgery 112:327; Ildstad et al., 1992, Transplantation 53:815; and Ildstad et al., 1992, J. Exp. Med. 175:147). Additionally, the use of human anti-xenograft, anti-idiotypic antibodies has also been proposed as a means by which to inhibit acute complement-mediated cytotoxicity (see U.S. Pat. No. 5,560,911,). To date, no one has successfully been able to achieve clinically significant attenuation of xenograft rejection in vivo, and it is unlikely that these other approaches will succeed in the near future.
2.4. RETARGETING OF HOST EFFECTOR MECHANISMS
Methods of retargeting host effector mechanisms to targets of therapeutic interest using bispecific agents have been reported in the art (see e.g., Meeting Report of the Second International Conference on Bispecific Antibodies and Targeted Cellular Toxicity, February 1991, Immunol. Today, 12(2):51-54). At the most basic level, any therapeutic antibody can be described as a bispecific agent that retargets host defense mechanisms to a chosen target. The antigen-binding "front" end of an antibody binds the antigenic epitope on a tumor cell, for example, and the Fc "tail" serves to attract and deliver host effector mechanisms, specifically complement or cells that possess receptors for the tail region of the antibody. These receptors, FcR, come in several varieties which bind different antibody populations and are expressed on different cell types (macrophages, neutrophils, natural killer, or NK cells, etc.). The host effector mechanisms are the ones that do the damage: complement forms a "membrane attack complex" comprised of components C5b-C9, which lyses the target cell, while FcR.sup.+ cells destroy target cells by either phagocytosis or by perforation of their membrane with lytic molecules (e.g., perforin, granzyme). Many strategies for retargeting cytotoxic cells have involved the use of bispecific conjugates of antibodies in which one antibody is directed against the cytotoxic cell receptor involved in lysis, while the second antibody is directed against a target cell structure, such as, for example, a tumor or viral antigen (see e.g., Donohue et al, 1990, Cancer Res. 50:6508-6514; Van Dijk et al., 1989, Int. J. Cancer, 44:738-743; and Segal et al., 1988, Princess Takamatsu Symp. 19:323-331). The administration of chemically cross-linked bispecific monoclonal antibodies reacting with CD3 on T-cells and with cell-surface antigens selectively expressed by tumor cells has been shown to target T-lymphocytes to neoplastic cells and to significantly decrease the growth of an established tumor in vivo (Garrido et al., 1990, Canc. Res. 50:4227-4232).
Recently, Pouletty has described a conjugate for inactivating target cells in a mammalian host which consists of a ligand for the target cell and a component that binds an endogenous cytotoxic effector system (European patent No. EPO 510949, issued Jan. 22, 1997). Pouletty further discloses methods for using these compounds to inactivate a target cell. Pouletty also describes the use of saccharides, such as the blood group A-trisaccharide antigen, as the effector-binding component of the conjugate and discloses the in vitro lysis of CTL-L2 lymphocytes after incubation with an IL2-blood group A conjugate and human serum containing anti-blood group A antibodies. However, Pouletty does not describe or suggest the use of .alpha.Gal oligosaccharides as the effector binding component of the conjugate. Nor does Pouletty describe or suggest harnessing the pre-existing anti-.alpha.Gal antibodies in the human serum as an effector agent for complement-mediated lytic attack. Indeed, Pouletty does not even disclose whether the blood group A antigen effector-binding component of the conjugate is effective in binding the endogenous effector system to form a cell inactivating complex in vivo.
Recently, Lussow et al. have described using an IL2-fluorescein conjugate to target anti-fluorescein antibodies to activated T cells, and thereby deplete the targeted cells in vivo (1996, Transplantation Proc., 28:571-572). Lussow et al. disclose that a IL2-fluorescein conjugate component ratio of 1:1 is critical for preventing clearance of this conjugate from the circulation. While Lussow et al. suggest using the .alpha.Gal epitope as the effector binding component of the conjugate to harness the hyperactive rejection response and redirect this response to desired targets, this reference does not teach whether any oligosaccharides, let alone oligosaccharides containing .alpha.Gal epitopes, can actually bind an endogenous effector system in vivo. Further, as discussed above, anti-.alpha.Gal antibodies are known to bind to .alpha.Gal oligosaccharides with relatively low affinity and it is doubtful that a monovalent .alpha.Gal oligosaccharide would bind anti-.alpha.Gal antibodies in vivo (see e.g., Parker et al., 1994, J. Immunology 153:3791-3803; Parker et al., 1995, Transplant Immunology 3:181-191). Accordingly, Lussow et al., does not provide a reasonable expectation that .alpha.Gal oligosaccharide-cell ligand conjugates could successfully be used to effectuate complement-mediated lytic attack of targeted cells in vivo.
Citation of a reference hereinabove shall not be construed as an admission that such reference is prior art to he present invention.